Sterilization of self-assembling peptides by irradiation

ABSTRACT

Gamma ray and e-beam irradiation provided efficient sterilization of certain self-assembling peptides (including RADA16 in solution) without substantial degradation of the major peptide, while, e.g., another self-assembly peptide, QLEL12 was significantly degraded following irradiation. Irradiation sterilization enhances the rheological property of, for example, RADA16 hydrogel once applied to tissue at a physiological pH. The rheological property increase can result in higher efficacy in a variety of biomedical applications.

CONTINUITY

This application is a divisional of U.S. patent application Ser. No.17/217,911, filed Mar. 30, 2021, which claims priority to U.S.provisional application No. 63/002,882, filed Mar. 31, 2020.

SEQUENCE LISTING

The instant application contains a XML Sequence Listing which is beingsubmitted electronically and is hereby incorporated by reference in itsentirety. Said Sequence Listing, created on Jan. 27, 2023 (EST), isnamed 3DM-20-01-IRR_US03-SLA.xml and is 32,737 bytes in size.

FIELD OF INVENTION

This invention relates to sterilization of certain medical gels, morespecifically, gels containing so-called self-assembling peptides.

BACKGROUND OF THE INVENTION

Self-assembling peptides (sometimes abbreviated as “SAPs”) are a type ofpeptides which assemble spontaneously into highly organizednanostructures when placed in aqueous environment and a chemical orphysical change in surrounding conditions occur. One other well-knownstructure is a nanofibrous biopolymer structure formed by naturalcollagen. A class of SAPs relevant for this invention consists ofalternating hydrophilic and hydrophobic amino acid residues capable offorming beta-sheets. They autonomously assemble into well-orderednanostructures in neutral water, while they can temporarily disassembleinto individual molecules when high shearing force is applied to them.SAPs can form a hydrogel (also known as SAP gels) depending on theirenvironment such as pH and/or osmolality; for example, they are capableof forming a hydrogel when they are placed in the body at near neutralpH. SAP gels have been previously described as being used for a varietyof medical applications, e.g., improved wound healing, inducement ofhomeostasis, reduction of adhesion in interior tissues, particularly, incontext of surgery; as temporary tissue-void matrix fillers,facilitating ingrowth of natural tissue into such a void. ParticularSAPs are described in U.S. Pat. Nos. 5,670,483; 5,955,343; 9,724,448;10,596,225 and Int'l Pat. Appln. Pub. WO2014/136081; and foreignequivalents thereof.

Sterilization is a very important step in the manufacturing process formost biomaterials, including for self-assembling peptide solutions.PuraStat® (RADA16=Ac-RADARADARADARADA-NH₂=SEQ ID NO:1; about 2.5%) iscustomarily filtered for sterilization (see, e.g., Int'l. Pat. Appln.Pub. No. WO 2014/008400); however, PuraStat®'s viscosity at higherconcentrations is the main obstacle to its filtration, which, inaddition to losses in the tubing, results in substantial peptide losses.In this method for sterilizing, a solution of SAPs is forced through aporous filter, wherein the sterilizing filter has an average pore sizeof 0.22 μm (US Pat. Appln. No. 2015/019735). As another method, somethermally stable self-assembling peptide solutions can be sterilized byautoclaving treatment at about 121° C. for about 25 minutes (U.S. patentapplication Ser. No. 10/369,237).

Furthermore, an additional ethylene oxide sterilization step is requiredfor the outer part of PuraStat® products. And, it was discovered thatautoclaving cannot be used for some SAPs, such as RADA16 (SEQ ID NO:1)in solution, because of its complete thermal degradation (US Pat. Appln.Pub. No. 2017/0202986). Thus, another new sterilization method has beenneeded to reduce losses, particularly, for RADA16 (SEQ ID NO:1) andother SAPs.

Gamma irradiation sterilization of self-assembling peptides includingRADA16 (SEQ ID NO:1) was described in passing in a prior publication (USPat. Appln. Pub. No. 2016/0317607; at paragraph [0052]); but no specificconditions or actual effect of irradiation on the structure andproperties of self-assembling peptides, including RADA16 (SEQ ID NO:1),have been described so far. In fact, peptide structure can be changed bythe reactive radical species generated by irradiation such as gamma ray,X-ray, and e-beam. Such a structural change can be governed by multiplefactors including the amino acid composition, reactive residue positionand possibly the conformation acquired by each macromolecule (Vieira Ret al, Biol. Pharm. Bull. 2013, 36(4) 664-675). For example, regardlessof the different sequences of the peptides in that article, all thetested nine peptides showed a progressive degradation by gamma rayirradiation up to 15 kGy. Considering that even higher doses ofirradiation than 15 kGy may be required for sterilization processes,numerous peptides cannot be sterilized with gamma ray irradiationwithout substantial degradation. It is well known that the side chainsof aromatic amino acids (i.e., Phenylalanine (F), Tyrosine (Y),Tryptophan (W), Histidine (H), and Proline (P)) and sulfur-containingamino acids (i.e., Cysteine (C)) are especially weak to attack byreactive radical species (Annu. Rev. Biochem., 62, 797-821 (1993) andVieira R et al, Biol. Pharm. Bull. 2013, 36(4) 664-675). By way ofbackground, the followings SAPs, RADA16 (Ac-RADARADARADARADA-NH₂ (SEQ IDNO:1)), KLD12 (Ac-KLDLKLDLKLDL-NH₂ (SEQ ID NO:2)), and IEIK13(Ac-IEIKIEIKIEIKI-NH₂ (SEQ ID NO:3)), do not include aromatic aminoacids or sulfur-containing amino acids.

On the other hand, irradiation sterilization can also affect thesecondary structure of self-assembling peptides and their fibrousstructure. As mentioned, RADA16 (SEQ ID NO:1), KLD12 (SEQ ID NO:2) andIEIK13 (SEQ ID NO:3) have beta-sheet conformation, and these moleculesself-assemble to form an ordered nanofibrous structure. The irradiationsterilization process may potentially change the secondary structure andthe nanofiber structure of peptide, which could then result in theundesirable change of its rheological properties.

Thus, there exists a need for new sterilization methods that workadvantageously with self-assembling peptides. For the reasons citedabove, sterilization by irradiation has been avoided so far.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the appearance of PuraStat® (RADA16 (SEQ ID NO:1) 2.5%)and IEIK13 (SEQ ID NO:3) 1.3% before gamma-irradiation, FIG. 1B showsthe appearance of PuraStat® (RADA16 (SEQ ID NO:1) 2.5%) and IEIK13 (SEQID NO:3) 1.3% after gamma-irradiation at 28 kGy.

FIG. 2A shows the mass spectrum of PuraStat® (RADA16 (SEQ ID NO:1) 2.5%)before gamma irradiation. FIG. 2B shows the mass spectrum of PuraStat®(RADA16 (SEQ ID NO:1) 2.5%) after gamma irradiation at 40 kGy. FIG. 2Cshows the mass spectrum of PuraStat® (RADA16 (SEQ ID NO:1) 2.5%) afterautoclaving at 121° C. for 20 min.

FIG. 3A shows the mass spectrum of PuraStat® (RADA16 (SEQ ID NO:1) 2.5%)after X-ray irradiation at 25 kGy. FIG. 3B shows the mass spectrum ofPuraStat® (RADA16 (SEQ ID NO:1) 2.5%) after X-ray irradiation at 40 kGy.

FIG. 4A shows the mass spectrum of PuraStat® (RADA16 (SEQ ID NO:1) 2.5%)after e-beam irradiation at 25 kGy. FIG. 4B shows the mass spectrum ofPuraStat® (RADA16 (SEQ ID NO:1) 2.5%) after e-beam irradiation at 40kGy.

FIG. 5A shows the mass spectrum of IEIK13 (SEQ ID NO:3) 1.3% beforeirradiation; FIG. 5B shows the mass spectrum of IEIK13 (SEQ ID NO:3)1.3% after gamma irradiation at 40 kGy; FIG. 5C shows the mass spectrumof IEIK13 (SEQ ID NO:3) 1.3% after X-ray irradiation at 25 kGy; FIG. 5Dshows the mass spectrum of IEIK13 (SEQ ID NO:3) 1.3% after X-rayirradiation at 40 kGy; FIG. 5E shows the mass spectrum of IEIK13 (SEQ IDNO:3) 1.3% after e-beam irradiation at 25 kGy; and FIG. 5F shows themass spectrum of IEIK13 (SEQ ID NO:3) 1.3% after e-beam irradiation at40 kGy.

FIG. 6A shows the mass spectrum of QLEL12 (SEQ ID NO:4) 0.15% beforeirradiation. FIG. 6B shows the mass spectrum of QLEL12 (SEQ ID NO:4)0.15% after gamma irradiation at 23 kGy. FIG. 6C shows the mass spectrumof QLEL12 (SEQ ID NO:4) 0.15% after X-ray irradiation at 25 kGy. FIG. 6Dshows the mass spectrum of QLEL12 (SEQ ID NO:4) 0.15% after X-rayirradiation at 40 kGy. FIG. 6E shows the mass spectrum of QLEL12 (SEQ IDNO:4) 0.15% after e-beam irradiation at 25 kGy. FIG. 6F shows the massspectrum of QLEL12 (SEQ ID NO:4) 0.15% after e-beam irradiation at 40kGy.

FIG. 7 shows results of a frequency test of PuraStat® (RADA16 (SEQ IDNO:1) 2.5%) with gamma irradiation at 0.1% of strain with 40 mmcone-plate (N=3, bars represent SD).

FIG. 8 shows results of a frequency test of PuraStat® (RADA16 (SEQ IDNO:1) 2.5%) with gamma irradiation after gelation at 0.1% of strain with40 mm cone-plate. Samples were treated with DMEM for 20 min (N=3, barsrepresent SD).

FIG. 9 shows results of a thixotropic test of PuraStat® (RADA16 (SEQ IDNO:1) 2.5%) with gamma irradiation at 1 Hz of frequency and 0.1% ofstrain with 40 mm cone-plate. Initial storage modulus (G′) was measuredfor 1 min before 1st shearing at 1000 s⁻¹. After the 1st shearing for 1minute, G′ recovery was recorded for 1 hour to exhibit the thixotropicbehavior of PuraStat®. This test was duplicated.

SUMMARY OF THE INVENTION

In the present disclosure, the effect of sterilization by irradiation onself-assembling peptide solutions was evaluated, specifically, with thefollowing peptides: PuraStat® (Ac-RADARADARADARADA-NH₂, RADA16 (SEQ IDNO:1), IEIK13 (Ac-IEIKIEIKIEIKI-NH₂ (SEQ ID NO:3)), and QLEL12(Ac-QLELQLELQLEL-NH₂ (SEQ ID NO:4)). It was unexpectedly found thatgamma-irradiation sterilization enhanced the rheological properties ofcertain self-assembling peptide solutions and hydrogels without theanticipated noteworthy degradation, while certain other peptides showedthe expected significant degradation and viscosity drop after gammairradiation (i.e., unlike RADA16 (SEQ ID NO:1) and IEIK13 (SEQ IDNO:3)), while, e.g., another self-assembling peptide, QLEL12(Ac-QLELQLELQLEL-NH₂ (SEQ ID NO:4)) was significantly degraded followingirradiation. This invention is further based on the prophetic findingthat the same result can be expected with other similar irradiationsterilization methods including, specifically, X-ray and e-beam, atleast for the respective peptides. Thus, in some embodiments, the methodof sterilizing a self-assembling peptide solution comprises:

-   -   a) placing one or more containers with a solution of        self-assembling peptide into an irradiation machine, said        self-assembling peptide capable of forming a hydrogel when        applied to a biological tissue at about neutral pH; and    -   b) exposing the container to gamma ray, X-ray and/or e-beam        irradiation at a predetermined dose so that the peptide solution        is sterilized without substantial degradation of the peptide        while its desired biological and/or rheological property(ies)        is/are maintained at the same level or improved.

In some embodiments, the peptides are selected from the group consistingof RADA16 (SEQ ID NO:1), KLD12 (SEQ ID NO:2), and IEIK13 (SEQ ID NO:3).In related embodiments, such peptides are exposed to the dose is 15-50kGy, preferably 15-40 kGy, more preferably, to a minimum dose resultingin a desired sterility assurance level (SAL), without substantialdegradation and/or substantial negative change in biological propertiesof these peptides. In some embodiments, the peptide solution isirradiated by gamma-rays, X-rays, or e-beam. In some embodiments, theover-all degradation of the total peptides in solution after irradiationdoes not exceed 20%, more preferably, 10%, most preferably 5%, of theamount of peptides prior to irradiation. In some embodiments, thedesired biological or physical property(ies) is/are selected from thegroup consisting of: hemostatic, anti-adhesion, prevention ofre-bleeding, anti-stenosis, tissue occlusion, storage modulus (e.g., insome embodiments, the storage modulus of the gelled solution isincreased at least by 10%, at least by 15% or at least by 20%post-irradiation, and viscosity, and tissue void filling property aremaintained within acceptable or improved parameters after irradiation.In some embodiments, irradiation dose achieves sterility assurance level(SAL) of at least 10⁻⁵, preferably 10⁻⁶, or less. In other embodiments,using the bioburden testing described below in the example, theacceptable level of contamination of the peptide solutionpre-irradiation is 1000, 500, 100, 15, 10, 9, 5, 2, 1.5, 1 CFU, or less.In some embodiments, the concentration of the degradation products ofthe intact (“major” or “full-length”) peptide in the solutionpost-irradiation ranges from 0.1% to 5%. In some embodiments, the pH ofthe peptide solution post-irradiation ranges from about 1.8 to 3.5. Insome embodiments, the solution container is a plastic syringe, with orwithout an adapter nozzle. In such embodiments, care is taken to ensurethat the plastic and rubber parts of the packaging also maintain theirdesired physical properties. While some yellowing of the plasticsyringes may be expected and is normal, any rubberized material mustpreserve its e plastic properties at an acceptable level.

In some embodiments, the gelled solution is further subjected tosheering to reduce or restore its storage modulus.

Thus, the invention provides a sterilization method for self-assemblingpeptides. According to the methods of the invention, in someembodiments, the solution is further applied to a biological tissue, forexample, during surgery, or after trauma involving bleeding.

DETAILED DESCRIPTION OF THE INVENTION

As stated above, in the present disclosure, the effect of sterilizationby irradiation on self-assembling peptide solutions was evaluated,specifically, with the following peptides: PuraStat®(Ac-RADARADARADARADA-NH₂ (SEQ ID NO:1); RADA16), IEIK13(Ac-IEIKIEIKIEIKI-NH₂ (SEQ ID NO:3)), and QLEL12 (Ac-QLELQLELQLEL-NH₂(SEQ ID NO:4)). In some embodiments, the method of sterilizing aself-assembling peptide solution comprises:

-   -   a) placing one or more containers with a solution of        self-assembling peptide into an irradiation machine, said        self-assembling peptide capable of forming a hydrogel when        applied to a biological tissue (e.g., in situ) at about neutral        pH; and    -   b) exposing the container to gamma ray, X-ray and/or e-beam        irradiation at a predetermined dose so that the peptide solution        is sterilized to a pre-determined Sterility Assurance Level        (SAL) without substantial degradation of the peptide while its        desired biological and/or physical property(ies) is/are        maintained substantially at the same level or improved.

It was unexpectedly found that gamma-irradiation sterilization enhancedthe rheological properties of certain self-assembling peptide solutionsand hydrogels without causing any noteworthy degradation, while certainother peptides showed significant degradation and viscosity drop aftergamma irradiation, i.e., unlike RADA16 (SEQ ID NO:1) and IEIK13 (SEQ IDNO:3), another self-assembling peptide, QLEL12 (Ac-QLELQLELQLEL-NH₂ (SEQID NO:4)) was significantly degraded following irradiation.

This invention is further based on the prophetic finding that the sameresult can be expected with other similar irradiation sterilizationmethods including, specifically, X-ray and e-beam, at least for therespective peptides. In preferred embodiments, the composition andmethods of the invention maintain or improve the desired biologicalproperty(ies) such as hemostatic, anti-adhesion, prevention ofre-bleeding, anti-stenosis, tissue occlusion, storage modulus,viscosity, and tissue void filling property, etc. For example, in someembodiments, the storage modulus in increased by at least 5%, 10%, 15%,20%, or more. If such increase is undesirable for certain applications,the gel can be thinned further by dilution or sheering by methods knownin the art, turning it into the solution or otherwise reducing itsstorage modulus. In certain embodiments, the irradiated solution of SAPsremains clear and viscous.

In some embodiments, the SAPs comprise a sequence of amino acid residuesconforming to one or more of Formulas I-IV:((Xaa^(neu)−Xaa⁺)_(x)(Xaa^(neu)−Xaa⁻)_(y))_(n)  (I)((Xaa^(neu)−Xaa⁻)_(x)(Xaa^(neu)−Xaa⁺)_(y))_(n)  (II)((Xaa⁺−Xaa^(neu))_(x)(Xaa⁻−Xaa^(neu))_(y))_(n)  (III)((Xaa⁻−Xaa^(neu))_(x)(Xaa⁺−Xaa^(neu))_(y))_(n)  (IV)Xaa^(neu) represents an amino acid residue having a neutral charge; Xaa⁺represents an amino acid residue having a positive charge; Xaa⁻represents an amino acid residue having a negative charge; x and y areintegers having a value of 1, 2, 3, or 4, independently; and n is aninteger having a value of 1-5.

In some embodiments, the SAPs further comprise an amino acid sequencethat interacts with the extracellular matrix, wherein the amino acidsequence anchors the SAPs to the extracellular matrix.

In some embodiments, the amino acid residues in the SAPs can benaturally occurring or non-naturally occurring amino acid residues.Naturally occurring amino acids can include amino acid residues encodedby the standard genetic code as well as non-standard amino acids (e.g.,amino acids having the D-configuration instead of the L-configuration),as well as those amino acids that can be formed by modifications ofstandard amino acids (e.g., pyrolysine or selenocysteine). Suitablenon-naturally occurring amino acids include, but are not limited to,D-alloisoleucine(2R,3S)-2-amino-3-methylpentanoic acid, L-cyclopentylglycine (S)-2-amino-2-cyclopentyl acetic acid.

In other embodiments, another class of materials that can self-assembleare peptidomimetics. Peptidomimetics, as used herein, refers tomolecules which mimic peptide structure. Peptidomimetics have generalfeatures analogous to their parent structures, polypeptides, such asamphiphilicity. Examples of such peptidomimetic materials are describedin Moore et al., Chem. Rev. 101(12), 3893-4012 (2001). Thepeptidomimetic materials can be classified into four categories:α-peptides, β-peptides, γ-peptides, and δ-peptides. Copolymers of thesepeptides can also be used. Examples of α-peptide peptidomimeticsinclude, but are not limited to, N,N′-linked oligoureas,oligopyrrolinones, oxazolidin-2-ones, azatides and azapeptides. Examplesof β-peptides include, but are not limited to, β-peptide foldamers,α-aminoxy acids, sulfur-containing β-peptide analogues, and hydrazinopeptides. Examples of γ-peptides include, but are not limited to,γ-peptide foldamers, oligoureas, oligocarbamates, and phosphodiesters.Examples of δ-peptides include, but are not limited to, alkene-basedδ-amino acids and carbopeptoids, such as pyranose-based carbopeptoidsand furanose-based carbopeptoids.

In certain embodiments, the SAP is AC5®, AC5-V®, AC5-G™ or TK45, alsoknown as AC1, made by Arch Therapeutics, Inc. (seewww.archtherapeutics.com).

In some embodiments, the SAP solution is contained in “storage and/ordrug delivery system”, such as, for example, storage and/or deliverysystems suitable for peptide compositions described herein, for example,vials, bottles, beakers, bags, syringes, ampules, cartridges,reservoirs, or LYO-JECTS®. Storage and/or delivery systems need not beone in the same and can be separate. In specific embodiments, SAPs areprovided in a plastic syringe, containing about 10 ml, about 7.5 ml,about 5, about 2.5, about 1, or about 0.5 ml of a SAP solution. Incertain embodiments, the plastics (e.g., plastic syringe) may acquire ayellowish tint after irradiation, which is normal, and does not affectbiomedical characteristics of the therein contained SAP

Solution

In some embodiments, such storage and delivery system may furthercontain a “nozzle” which refers to a generally thin, cylindrical object,often with a narrow end and a wide end, which is adapted for fixing ontoa delivery device described herein. In some embodiments, the terms“nozzle” and “cannula” are used interchangeably. Nozzles are composed oftwo connection points or ends, a first connection point or end toconnect to a delivery system (e.g., a syringe) and a second connectionpoint which may serve as the point where delivery of pharmaceuticalcomposition is administered or as a point to connect to a secondarydevice (e.g., a catheter).

Thus, the invention provides methods of making sterilized solutions ofthe self-assembling peptides. The invention also provides use of suchsterilized solutions applied to a biological tissue, e.g., in situ, forexample, during surgery or after trauma involving bleeding, or use ofso-sterilized solutions in treatment or prevention of other diseases orconditions; for example, as described in as described in Int'l Pat.Appln. WO2014/133027, or as described in US Pat. Appln. Pub. Nos.2011/02101541 and occlude a site of tissue damage; or as described USPat. Appln. Pub. No. 2016/0287744 for vascular embolization; or asdescribed in U.S. patent application Ser. No. 16/085,803 for occlusionof cerebrospinal fluid leakage, or as described in Int'l Pat. Pub.Appln. No. WO2013/133414 as mucosa elevating agent; or as described inInt'l Pat. Pub. Appln. No. WO2014/141160 for bile leakage occlusion; oras described in U.S. patent application Ser. No. 16/885,753 foranti-adhesion of tissues; or as described in U.S. patent applicationSer. No. 16/085,804 for pancreatic fistula occlusion; or as described inU.S. patent application Ser. No. 15/124,639 for bronchial obstruction;or as described in Int'l Pat. Appln. Pub. No. WO2015/138,478 fortreating pulmonary bulla collapse; or as described in Int'l Pat. Appln.Pub. No. WO2015/019,738 for treatment of pulmonary leakage; or asdescribed in Int'l Pat. Appln. Pub. No. WO2015/196020 for filling dentalbone voids; or as described US Pat. Appln. Pub. No. 2017/0128622 forfilling bone voids; or as described in U.S. patent application Ser. No.16/312,878 for the prevention of esophageal structure after endoscopicdissection, and foreign equivalents of any of the aforementionedpublications, and other methods known in the art. Accordingly, in someembodiments, the invention provides the use of a sterilized solution ofthe self-assembling peptide for treating or preventing an aforementioneddisease or condition, wherein the sterilized solution is obtained by themethods of the invention. In certain such embodiments, theself-assembling peptide solution exhibits a post-irradiation massspectrometric (MS) profile substantially as shown in correspondingpost-irradiation profiles of FIGS. 2-6 and/or as described in theExamples. For example, in the case of PuraStat®, the additional majorM_(z) peaks at are observed at 836/1670, 1100, and 1513 m/z.

An average bioburden <1,000 CFU is the typical sterility of PuraStat®before sterilization. In this case, to achieve a sterility assurancelevel, SAL, of 10⁻⁶, the range of irradiation dose should be between 25kGy and 40 kGy. With gamma and X-ray methods, about up to 20% of theover-all peptides may degrade during sterilization process. With e-beammethod, about up to 10% of the over-all peptides may degrade duringsterilization process. PuraStat® rheology increases after sterilizationby gamma-ray, or X-ray, which can alter its hemostatic efficacypositively or negatively. However, PuraStat® rheology does not changeafter e-beam sterilization.

In certain embodiments, using the bioburden testing described below inthe Examples, the acceptable level of contamination of the peptidesolution pre-irradiation may be <1000, <500, <100, <15, <10, <9, <5, <2,<1.5, <1 CFU, or less per product unit. The preferred bioburdenpre-sterilization is <9 CFU. Thus, under 9 CFU, the range of irradiationdose can be selected between 15 kGy and 24 kGy. While the low end of theirradiation dose range may be determined by the bioburden of thepre-irradiated product, the high end of the range is chosen based on theconfiguration and choice of the irradiator machine and set at a minimumrequired to achieve desirable sterility assurance level (SAL).

With gamma and X-ray methods, up to 10% of the peptides may degradeduring sterilization process. With e-beam method, the peptides may notsignificantly degrade during sterilization process. Even in this case,PuraStat® rheology increases after gamma-ray and X-ray sterilization,which may somewhat change its hemostatic efficacy positively ornegatively. Thus, PuraStat® rheology does not change after e-beamsterilization and thus may be preferred, if no or minimal change inrheological properties and its hemostatic properties is desirable.Therefore, e-beam irradiation may be particularly preferred forPuraStat®.

In some embodiments, the concentration of degraded full-length peptide(“major peptide”) in the solution post-irradiation ranges from 0.1% to5%, 0.1% to 4%, 0.1% to 3%, 0.1% to 2.5%, 0.1% to 2%, or 0.1% or 1.5% orless. In some embodiments, for RADA16 (SEQ ID NO:1), KLD12 (SEQ IDNO:2), or IEIK13 (SEQ ID NO:3) solution irradiated with a total dose,which may depend on its pre-irradiation bioburden, including, forexample, 15-50 kGy, 25 kGy+/−15 kGy, 40 kGy-10 kGy, 35 kGy-10 kGy, 30kGy-10 kGy, 15 kGy-24 KGy, 25 kGy-10 kGy, 20 kGy-10 kGy, 10 kGy-15 kGy,and 12 kGy-14 kGy. Doses around 12-14 kGy appear to be optimal for gammairradiation, however, the peptide solution may also be irradiated withsimilar doses with X-ray or e-beam.

In some embodiments, the “storage and/or drug delivery system”, forexample, a plastic syringe contained in a blister pack, is irradiated inbatches of at least: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 units ormore at a time. Generally, it is preferred that the total exposure ofany sample in an irradiated batch does not exceed 100 kGy, morepreferably 60 kGy, most preferably, 50 kGy, or less as described herein.

In some embodiments, the major peptide's degradation (also referred toas “full-length peptide's degradation”) after the irradiation does notexceed 20% of the amount of the peptide prior to the irradiation and,preferably, does not exceed 18%, 16%, 14%, 12%, 10%, 8%, 5%, 3%, or 1%.In some embodiments, the total dose is achieved over a period of timesufficient to achieve necessary sterility of the solution. For example,the dose of 40 kGy can be delivered by radiation intensity 6.3 kGy/hrfor about 6 hrs and 21 min. Other combinations can be found inExample 1. In some embodiments, a constant dose was delivered over anumber of hours, e.g., about 10, about 9, about 8, about 7, about 6,about 5, about 4, about 3, or about 2 hours.

In some embodiments, samples are irradiated with gamma ray doses, forexample, of about 23, about 25, about 28 or about 40 kGy, or other dosesindicated above with an instrument, for example, such as Gammacell 220®High Dose Rate Co-60 Irradiator in VPTrad (www.vptrad.com; MDS Nordion,Ottawa, Canada).

In other embodiments, samples are irradiated with X-ray with doses ofabout 25 kGy to about 40 kGy, or the doses as indicated above, with aMevex accelerator (with the following settings: 10 MeV, 20 kW).

In general, X-ray frequency range is 3×10¹⁶-3×10¹⁹ Hz, while thefrequency range of gamma-rays is 3×10¹⁹ or higher.

In other embodiments, samples are irradiated with e-beam at about 25 toabout 40 kGy with a Mevex accelerator (with the following settings: 10MeV, 20 kW) (seemevex.com/linacs/10mev-system-e-beam-sterilization-for-medical-devices/).

In some embodiments, the desired biological and physical property(ies)is/are selected from the group consisting of: hemostatic, anti-adhesion,prevention of re-bleeding, anti-stenosis, tissue occlusion, storagemodulus, viscosity, tissue void filling property, mucosa elevation, andwound healing,

In preferred embodiments, the irradiation dose achieves a sterilityassurance level (SAL) of at least 10⁻⁵, 10⁻⁶, or less.

The invention also provides the sterilized solution of self-assemblingpeptide made by the methods described herein. Such comprising: applyingthe solution to a biological tissue, for example, during surgery orafter trauma involving bleeding, to a site with substantially neutralpH, resulting in gelling. In some embodiments, such site is internal,while in other embodiment the site can be external such surface sutures,cuts, or scrapes.

Other aspects of the invention would be apparent to those of skill inthe art based on the present description, including the Examples and theappended claims.

Example 1: Irradiation Conditions

Samples were irradiated with gamma rays at 23, 25, 28 and 40 kGy withVPTrad Gammacell 220® High Dose Rate Co-60 Irradiator. In someembodiments, the run dose rate and duration time for 40 kGy irradiationwere 6.30 kGy/hr and 6 hours 20 minutes 58 seconds, respectively. Inother embodiments, the run dose rate and duration time for 28 kGyirradiation were 4.40 kGy/hr and 6 hours 22 minutes 31 seconds,respectively. In other embodiments, the run dose rate and duration timefor 25 kGy irradiation were 6.58 kGy/hr and 3 hours 47 minutes 42seconds, respectively. In other embodiments, the run dose rate andduration time for 23 kGy irradiation were 6.58 kGy/hr and 3 hours 29minutes 29 seconds, respectively.

In other embodiments, samples were irradiated with X-ray at 25 kGy and40 kGy with a Mevex accelerator (10 MeV, 20 kW).

In other embodiments, samples were irradiated with e-beam at 25 and 40kGy with a Mevex accelerator (10 MeV, 20 kW).

Example 2: HPLC Conditions

HPLC tests were performed to evaluate the major peptide content afterirradiation tests. An Agilent HPLC 1100 (Agilent Technologies) was usedfor this study. The column temperature was kept at 25° C.

For RADA16 (SEQ ID NO:1) samples, solvent A was water with 0.1% TFA andsolvent B was 80% acetonitrile with 0.1% TFA. Gradient of solvent B wascontrolled from 10% to 40% in 20 min and 40% for another 5 min at 25° C.Agilent Zorbax 300SB-C18 column (4.6 mm×250 mm, 5 m, 300 Å) was used forthis test. PuraStat® (RADA16 (SEQ ID NO:1) 2.5%) (40 mg) was mixed with10 μL of DH₂O and the mixture was vortexed. The mixture was furthermixed with 500 μL of formic acid and vortexed. This mixture was thenmixed with DH₂O (4,450 μL) and vortexed. 20 μL samples were injectedusing an Agilent autosampler.

For IEIK13 (SEQ ID NO:3) samples, solvent A was water with 0.1% TFA andsolvent B was 90% Acetonitrile with 0.1% TFA. The gradient of solvent Bwas controlled from 20% to 43% in 8 min, from 43% to 70% between 8 and9.5 min, and from 70% to 95% between 9.5 and 30 min, and 95% for another5 min at 25° C. An Agilent PLRP-S column (4.6 mm×250 mm, 8 m, 300 Å) wasused for this test. A 1.3% solution of IEIK13 (SEQ ID NO:3) was dilutedwith water with 0.1% TFA to 0.075% and the mixture was vortexed. 35 μLsamples were injected using an Agilent autosampler.

For QLEL12 (SEQ ID NO:4) samples, solvent A was water and solvent B was80% acetonitrile. The gradient of solvent B was controlled from 20% to80% in 15 min and 80% for another 7 min at 25° C. An Agilent PLRP-Scolumn (4.6 mm×250 mm, 8 m, 300 Å) was used for this test. 0.15% w/v ofQLEL12 (SEQ ID NO:4) solution was diluted to 0.01% with DH₂O andvortexed. 50 μL samples were injected using an Agilent autosampler.

Example 3: Mass Spectrometry Conditions

Mass spectrometry tests were carried out to investigate the degradationof the peptides after irradiation sterilization. An Agilent LC/MSD iontrap mass spectrometer was used for this study. The sample solutionswere prepared as described above for the HPLC samples. Each sample wasinjected at 9 μL/min with a syringe pump. Each mass spectrum wasrecorded for 1 minute.

Example 4: Rheological Properties of Irradiated Self-Assembling Peptides

The rheological properties of samples were evaluated using a rheometer(DHR1, TA Instruments) with 40 mm cone and plate. Peptide solution (700μL) was placed on the rheometer plate and excess solution was gentlyremoved by a metal spatula. Measurements were performed after 2 minutesof relaxation time at 37° C.

Frequency tests were performed at 0.1% of strain from 0.1 Hz to 10 Hz.Frequency tests after gelation were performed under the same conditionsfor 20 minutes after 3 mL of DMEM was gently added around the cone andthe plate.

Thixotropic tests were carried out with the following method. A shearrate of 1000 s⁻¹ was applied for 1 minute to reset all rheologicalproperties. Then 1 Hz of frequency at 0.1% of strain was applied for 60minutes to record thixotropic behaviors. This sequence was repeated, andthe thixotropic properties were then analyzed.

Example 5: Appearance and pH

The appearance and pH of PuraStat® after gamma irradiation are shown inFIG. 1 and Table 1 (N=3).

TABLE 1 Appearance and pH of PuraStat ® (RADA16 (SEQ ID NO: 1) 2.5%)after gamma irradiation. Testing conditions Appearance pH PuraStat ®control Clear and viscous 2.2 PuraStat ® irradiated (gamma) at 23 kGyClear and viscous 2.3 PuraStat ® irradiated (gamma) at 25 kGy Clear andviscous 2.3 PuraStat ® irradiated (gamma) at 28 kGy Clear and viscous2.3 PuraStat ® irradiated (gamma) at 40 kGy Clear and viscous 2.3The appearance and pH of PuraStat® after gamma irradiation were onlyslightly altered. The pH of the PuraStat® control was 2.2. The pH ofPuraStat® after gamma irradiation at 23, 25, 28, and 40 kGy were 2.3.

The appearance and pH of IEIK13 (SEQ ID NO:3) 1.3% after gammairradiation are also shown in FIG. 1 and Table 2.

TABLE 2 Appearance and pH of IEIK13 (SEQ ID NO: 3) 1.3%, KLD12 (SEQ IDNO: 2) 1.3% and QLEL12 (SEQ ID NO: 4) 0.15% after gamma irradiation. (N= 3) Testing conditions Appearance pH IEIK13 (SEQ ID NO: 3) controlClear and viscous 3.0 IEIK13 (SEQ ID NO: 3) irradiated Clear and viscous3.0 (gamma) at 28 kGy IEIK13 (SEQ ID NO: 3) irradiated Clear and viscous3.0 (gamma) at 40 kGy KLD12 (SEQ ID NO: 2) control Clear and viscous 2.2KLD12 (SEQ ID NO: 2) irradiated Clear and viscous 2.2 (gamma) at 40 kGyQLEL12 (SEQ ID NO: 4) control Clear and viscous 7.0 QLEL12 (SEQ ID NO:4) irradiated Clear and watery 6.7 (gamma) at 23 kGy QLEL12 (SEQ ID NO:4) irradiated Clear and watery 6.5 (gamma) at 25 kGy QLEL12 (SEQ ID NO:4) irradiated Clear and watery 5.9 (gamma) at 40 kGy

The appearance and pH of IEIK13 (SEQ ID NO:3) (1.3%) after gammairradiation were not changed. The pH of IEIK13 (SEQ ID NO:3) control was3.0. The pH of IEIK13 (SEQ ID NO:3) (1.3%) after gamma irradiation at 28kGy and at 40 kGy was 3.0.

The appearance and pH of KLD12 (SEQ ID NO:2) (1.3%) and QLEL12 (SEQ IDNO:4) (0.15%) after gamma irradiation were also shown in Table 2. Theappearance and pH of KLD12 (SEQ ID NO:2) (1.3%) after gamma irradiationwere not changed. The pH of KLD12 (SEQ ID NO:2) control was 2.2. The pHof KLD12 (SEQ ID NO:2) (1.3%) after gamma irradiation at 40 kGy were2.2.

However, the appearance and pH of QLEL12 (SEQ ID NO:4) after gammairradiation were significantly altered. The pH of the QLEL12 (SEQ IDNO:4) control was 7.0. The pH of QLEL12 (SEQ ID NO:4) after gammairradiation at 23, 25 and 40 kGy were 6.7, 6.5 and 5.9, respectively,and the samples became watery after gamma irradiation.

The appearance and pH of PuraStat® after X-ray and e-beam irradiationare also shown in Table 3 (N=3).

TABLE 3 Appearance and pH of PuraStat ® (RADA16 (SEQ ID NO: 1) 2.5%)after X-ray and e-beam irradiation test. (N = 3) Testing conditionsAppearance pH PuraStat ® control Clear and viscous 2.2 PuraStat ®irradiated (X-ray) at 25 kGy Clear and viscous 2.3 PuraStat ® irradiated(X-ray) at 40 kGy Clear and viscous 2.3 PuraStat ® irradiated (e-beam)at 25 kGy Clear and viscous 2.2 PuraStat ® irradiated (e-beam) at 40 kGyClear and viscous 2.2

The appearance and pH of PuraStat® after X-ray or e-beam irradiation wasonly slightly changed or remained unchanged. The pH of PuraStat® controlwas 2.2. The pH of PuraStat® after X-ray irradiation at 25 kGy and at 40kGy was 2.3. The pH of PuraStat® after e-beam irradiation at 25 kGy andat 40 kGy was 2.2.

The appearance and pH of IEIK13 (SEQ ID NO:3) 1.3% after X-ray or e-beamirradiation are also shown in Table 4.

TABLE 4 Appearance and pH of IEIK13 (SEQ ID NO: 3) 1.3% after X-ray ore-beam irradiation (N = 3). Testing conditions Appearance pH IEIK13 (SEQID NO: 3) control Clear and viscous 3.0 IEIK13 (SEQ ID NO: 3) irradiated(X-ray) Clear and viscous 3.0 at 25 kGy IEIK13 (SEQ ID NO: 3) irradiated(X-ray) Clear and viscous 3.0 at 40 kGy IEIK13 (SEQ ID NO: 3) irradiated(e-beam) Clear and viscous 3.0 at 25 kGy IEIK13 (SEQ ID NO: 3)irradiated (e-beam) Clear and viscous 3.0 at 40 kGy

The appearance and pH of IEIK13 (SEQ ID NO:3) 1.3% after X-ray or e-beamirradiation were not changed. The pH of IEIK13 (SEQ ID NO:3) 1.3%control was 3.0. The pH of IEIK13 (SEQ ID NO:3) 1.3% after X-ray ore-beam irradiation at 28 kGy and 40 kGy were 3.0.

The appearance and pH of KLD12 (SEQ ID NO:2) 1.3% after X-ray or e-beamirradiation are also shown in Table 5.

TABLE 5 Appearance and pH of KLD12 (SEQ ID NO: 2) 1.3% after X-ray ore-beam irradiation (N = 3). Testing conditions Appearance pH KLD12 (SEQID NO: 2) control Clear and viscous 2.2 KLD12 (SEQ ID NO: 2) irradiated(X-ray) Clear and viscous 2.2 at 25 kGy KLD12 (SEQ ID NO: 2) irradiated(X-ray) Clear and viscous 2.2 at 40 kGy KLD12 (SEQ ID NO: 2) irradiated(e-beam) Clear and viscous 2.2 at 25 kGy KLD12 (SEQ ID NO: 2) irradiated(e-beam) Clear and viscous 2.2 at 40 kGy

The appearance and pH of KLD12 (SEQ ID NO:2) 1.3% after X-ray or e-beamirradiation were not changed. The pH of KLD12 (SEQ ID NO:2) 1.3% controlwas 2.2. The pH of KLD12 (SEQ ID NO:2) 1.3% after X-ray or e-beamirradiation at 25 kGy and 40 kGy was 2.2.

The appearance and pH of QLEL12 (SEQ ID NO:4) 0.15% after X-ray ore-beam irradiation are also shown in Table 6.

TABLE 6 Appearance and pH of QLEL12 (SEQ ID NO: 4) 0.15% after X-ray ande-beam irradiation (N = 3). Testing conditions Appearance pH QLEL12 (SEQID NO: 4) control Clear and viscous 7.0 QLEL12 (SEQ ID NO: 4) irradiated(Gamma) Clear and watery 6.5 at 25 kGy QLEL12 (SEQ ID NO: 4) irradiated(Gamma) Clear and watery 5.9 at 40 kGy QLEL12 (SEQ ID NO: 4) irradiated(X-ray) Clear and watery 6.3 at 25 kGy QLEL12 (SEQ ID NO: 4) irradiated(X-ray) Clear and watery 5.9 at 40 kGy QLEL12 (SEQ ID NO: 4) irradiated(e-beam) Clear and watery 6.6 at 25 kGy QLEL12 (SEQ ID NO: 4) irradiated(e-beam) Clear and watery 6.5 at 40 kGy

The appearance of QLEL12 (SEQ ID NO:4) after gamma, X-ray or e-beamirradiation was significantly altered. The pH of the QLEL12 (SEQ IDNO:4) control was 7.0. The pH's of QLEL12 (SEQ ID NO:4) after gammairradiation at 25 and 40 kGy were 6.5 and 5.9, respectively, those afterX-ray irradiation at 25 and 40 kGy were 6.3 and 6.5, respectively, andthose after e-beam irradiation at 25 and 40 kGy were 6.6 and 6.5,respectively. QLEL12 (SEQ ID NO:4) solutions became watery after gamma,X-ray or e-beam irradiation.

In summary, RADA16 (SEQ ID NO:1), IEIK13 (SEQ ID NO:3) and KLD12 (SEQ IDNO:2) remained unchanged after gamma, X-ray and e-beam irradiation ataround 25˜40 kGy, but QLEL12 (SEQ ID NO:4) was changed. Therefore,unlike QLEL12 (SEQ ID NO:4), RADA16 (SEQ ID NO:1), IEIK13 (SEQ ID NO:3),and KLD12 (SEQ ID NO:2) can be sterilized using a gamma, X-ray ande-beam irradiation techniques.

Example 6: Characterization by HPLC and Mass Spectrometry

The HPLC tests for RADA16 (SEQ ID NO:1), IEIK13 (SEQ ID NO:3), andQLEL12 (SEQ ID NO:4) were performed before and after gamma, X-ray ande-beam irradiation, and the results for their major peptide content arelisted in Tables 7-10.

TABLE 7 HPLC test for major peptide content of PuraStat ® (RADA16 (SEQID NO: 1) 2.5%) before and after gamma- irradiation (N = 3, mean ± SD).Major peptide content Testing conditions (%) PuraStat ® control 78.3 ±1.7  PuraStat ® irradiated (gamma) at 23 kGy 75.5 ± 1.4* PuraStat ®irradiated (gamma) at 25 kGy 74.6 ± 0.7* PuraStat ® irradiated (gamma)at 28 kGy 68.5 ± 0.5* PuraStat ® irradiated (gamma) at 40 kGy 69.7 ±0.5* *denotes if significantly lower than the data of PuraStat ® control(if p < 0.05, two tailed Student's t-test).

TABLE 8 HPLC test for major peptide contents of PuraStat ® (RADA16 (SEQID NO: 1) 2.5%) before and after X-ray and e-beam irradiation (N = 3,mean ± SD). Major peptide content Testing conditions (%) PuraStat ®control 78.3 ± 1.7  PuraStat ® irradiated (X-ray) at 25 kGy 73.8 ± 1.7*PuraStat ® irradiated (X-ray) at 40 kGy 71.9 ± 0.8* PuraStat ®irradiated (e-beam) at 25 kGy 76.0 ± 0.9* PuraStat ® irradiated (e-beam)at 40 kGy 70.0 ± 2.4* *denotes if significantly lower than the data ofPuraStat ® control (if p < 0.05, two tailed Student's t-test).

TABLE 9 HPLC test for major peptide contents of IEIK13 (SEQ ID NO: 3)1.3% before and after gamma, X- ray, and e-beam irradiation (N = 3, tmean ± SD). Major peptide content Testing conditions (%) IEIK13 (SEQ IDNO: 3) control 99.6 ± 0.0 IEIK13 (SEQ ID NO: 3) irradiated (gamma) 99.8± 0.2 at 40 kGy IEIK13 (SEQ ID NO: 3) irradiated (X-ray) 99.8 ± 0.1 at25 kGy IEIK13 (SEQ ID NO: 3) irradiated (X-ray) 99.9 ± 0.1 at 40 kGyIEIK13 (SEQ ID NO: 3) irradiated (e-beam) 99.6 ± 0.2 at 25 kGy IEIK13(SEQ ID NO: 3) irradiated (e-beam) 99.8 ± 0.1 at 40 kGy

TABLE 10 HPLC test for major peptide contents of QLEL12 (SEQ ID NO: 4)0.15% before and after gamma, X- ray, and e-beam irradiation (N = 3,mean ± SD). Major peptide content Testing conditions (%) QLEL12 (SEQ IDNO: 4) control 90.5 ± 1.3  QLEL12 (SEQ ID NO: 4) irradiated (gamma) 40.7± 3.1* at 40 kGy QLEL12 (SEQ ID NO: 4) irradiated (X-ray) 57.0 ± 4.9* at25 kGy QLEL12 (SEQ ID NO: 4) irradiated (X-ray) 47.3 ± 1.3* at 40 kGyQLEL12 (SEQ ID NO: 4) irradiated (e-beam) 51.9 ± 7.3* at 25 kGy QLEL12(SEQ ID NO: 4) irradiated (e-beam) 55.6 ± 3.0* at 40 kGy *denotes ifsignificantly lower than the data of QLEL12 (SEQ ID NO: 4) control (if p< 0.05, two tailed Student's t-test).

The major peptide content of RADA16 (SEQ ID NO:1) control was 78.3%. Themajor peptide contents of RADA16 (SEQ ID NO:1) after gamma irradiationat 23, 25, 28 and 40 kGy were 75.5, 74.6, 68.5 and 69.7%, respectively.The major peptide contents of RADA16 (SEQ ID NO:1) after X-rayirradiation at 25 and 40 kGy were 73.8 and 71.9%, respectively. Themajor peptide contents of RADA16 (SEQ ID NO:1) after e-beam irradiationat 25 and 40 kGy were 76.0 and 70.0%, respectively.

The major peptide content of IEIK13 (SEQ ID NO:3) control was 99.6%. Themajor peptide content of IEIK13 (SEQ ID NO:3) after gamma irradiation at40 kGy was 99.8%. The major peptide contents of IEIK13 (SEQ ID NO:3)after X-ray irradiation at 25 and 40 kGy were 99.8 and 99.9%,respectively. The major peptide contents of IEIK13 (SEQ ID NO:3) aftere-beam irradiation at 25 and 40 kGy were 99.6 and 99.8%, respectively.

The major peptide content of QLEL12 (SEQ ID NO:4) control was 90.5%.However, QLEL12 (SEQ ID NO:4) showed significant decrease in its majorpeptide content after irradiation sterilization. The major peptidecontent of QLEL12 (SEQ ID NO:4) after gamma irradiation at 40 kGy was40.7%. The major peptide contents of QLEL12 (SEQ ID NO:4) after X-rayirradiation at 25 and 40 kGy were 57.0 and 47.3%, respectively. Themajor peptide contents of QLEL12 (SEQ ID NO:4) after e-beam irradiationat 25 and 40 kGy were 51.9 and 55.6%, respectively. These resultsdemonstrate that RADA16 (SEQ ID NO:1) and IEIK13 (SEQ ID NO:3) remainrelatively unchanged after gamma, X-ray and e-beam irradiation comparedto QLEL12 (SEQ ID NO:4).

The measured molecular weight of RADA16 (SEQ ID NO:1) was 1712, whichmatches its calculated molecular weight (FIG. 2 ). The mass spectrometryanalysis demonstrated that RADA16 (SEQ ID NO:1) was not degraded aftergamma, X-ray or e-beam irradiation (FIGS. 2-4 ). However, RADA16 (SEQ IDNO:1) completely degraded during autoclave treatment.

Irradiation is a cold temperature sterilization technique unlikeautoclaving. Although both sterilization techniques provide high energy(i.e., radiation and heat) to self-assembling peptides such as RADA16(SEQ ID NO:1) during sterilization, irradiation sterilization did notinduce substantial degradation of RADA16 (SEQ ID NO:1) molecules.

However, all gamma, X-ray, and e-beam irradiated PuraStat® mass spectraexhibited some peaks that were not observed before irradiation, while nonotable difference was observed among them. The summary of the massspectra is listed in Table 11.

TABLE 11Mass-spectrometry data of PuraStat ® after irradiation sterilization process.Mw at Mw at Mw at Control Irradiated Mz n = 1 n = 2 n = 3Estimated component PuraStat ®* PuraStat ®*  502  501 ARADA-NH₂ Yes Yes(SEQ ID NO: 5)  572 1712 Ac-(RADA)₄-NH₂ Yes Yes (SEQ ID NO: 1)  665 1328ARADARADARADA-NH₂ Yes Yes (SEQ ID NO: 6)  836 1670 (RADA)₄-NH₂ No Yes(SEQ ID NO: 7)  857 1712 Ac-(RADA)₄-NH₂ Yes Yes (SEQ ID NO: 1)  916  915ARADARADA-NH₂ Yes Yes (SEQ ID NO: 8) 1100 1100 ADARADARADA-NH₂ No Yes(SEQ ID NO: 9) 1143 1142 ARADARADARA Yes Yes (SEQ ID NO: 10) 1229 1228Ac-RADARADARAD Yes Yes (SEQ ID NO: 11) 1329 1328 ARADARADARADA-NH₂ YesYes (SEQ ID NO: 6) 1513 1513 ADARADARADARADA-NH₂ No Yes (SEQ ID NO: 12)1643 1642 Ac-RADARADARADARAD Yes Yes (SEQ ID NO: 13) 1670 1670(RADA)₄-NH₂ No Yes (SEQ ID NO: 7) 1713 1712 Ac-(RADA)₄-NH₂ Yes Yes(SEQ ID NO: 1) *: stored at 2-8° C. for about 4 years. *: all PuraStat® samples ware irradiated at 40 kGy (with gamma rays, X-rays, ande-beam)

The control PuraStat® showed M_(z) peaks at 572, 857, and 1713, whichare assigned to the major peptide, Ac-(RADA)₄-NH₂ (SEQ ID NO:1). ControlPuraStat® also exhibited other peaks at 502, 665/1329, 916, 1143, and1229 M_(z), which are estimated as ARADA-NH₂ (SEQ ID NO:5),ARADARADARADA-NH₂ (SEQ ID NO:6), ARADARADA-NH₂ (SEQ ID NO:8),ARADARADARA (SEQ ID NO:10), Ac-RADARADARAD (SEQ ID NO:11), respectively.

Table 12 shows the pattern of degradation of Ac-(RADA)₄-NH₂ (SEQ IDNO:1) when PuraStat® is stored at 2-8° C. for about 4 years. From thepattern, we found that degradation mainly occurs at the points between˜RAD and A˜.

On the other hand, the irradiated PuraStat® showed additional M_(z)peaks at 836/1670, 1100, and 1513, which are estimated as (RADA)₄-NH₂(SEQ ID NO:7), ADARADARADA-NH₂ (SEQ ID NO:9), and ADARADARADARADA-NH₂(SEQ ID NO:12), respectively.

Table 13 shows the additional pattern of degradation of Ac-(RADA)₄-NH₂(SEQ ID NO:1) when PuraStat® is sterilized with irradiation. Especially,the peaks at 836 and 1670 are ones of major additional peaks, whichrepresent RADARADARADARADA-NH₂ (SEQ ID NO:7). This means thatirradiation can cause additional degradation of PuraStat® at the pointbetween acetyl group (Ac) and RAD˜. The peaks at 1100 and 1513 are alsoones of major additional peaks, which represent ADARADARADA-NH₂ (SEQ IDNO:9), and ADARADARADARADA-NH₂ (SEQ ID NO:12). This means thatirradiation can cause additional degradation of PuraStat® at the pointbetween ˜R and AD˜.

TABLE 12 Pattern of degradation of Ac-(RADA)₄-NH₂ (SEQ ID NO: 1)(PuraStat ® was stored at 2-8° C. for about 4 years)Ac-RADARADARADARADA-NH₂ (SEQ ID NO: 1) (before degradation)Ac-RAD/ARADARADARADA-NH₂ (SEQ ID NO: 6)Ac-RADARAD (SEQ ID NO: 14)/ARADARADA-NH₂ (SEQ ID NO: 8)Ac-RADARADARAD (SEQ ID NO: 11)/ARADA-NH₂ (SEQ ID NO: 5)Ac-RAD/ARADARADARA (SEQ ID NO: 10)/DA-NH₂Ac-RADARADARADARAD (SEQ ID NO: 13)/A-NH₂

TABLE 13 Additional pattern of degradation of Ac-(RADA)₄-NH₂ (SEQ IDNO: 1) when PuraStat ® is sterilized with irradiationAc-RADARADARADARADA-NH₂ (SEQ ID NO: 1) (before degradation)Ac/RADARADARADARADA-NH₂ (SE ID NO: 7)Ac-R/ADARADARADARADA-NH₂ (SEQ ID NO: 12)Ac-RADAR (SEQ ID NO: 15)/ADARADARADA-NH₂ (SEQ ID NO: 9)

Also, the molecular weight of IEIK13 (SEQ ID NO:3) was measured at 1622,which matches its calculated molecular weight (FIG. 5 ). The mass specanalysis demonstrated that IEIK13 (SEQ ID NO:3) was only insubstantiallydegraded after gamma-ray, X-ray, and e-beam irradiation.

Furthermore, the molar mass of QLEL12 (SEQ ID NO:4) was measured at1506, which matches its calculated molar mass (FIG. 6 ). However, themass spec analysis demonstrated that QLEL12 (SEQ ID NO:4) wassignificantly degraded after gamma-ray, X-ray, and e-beam irradiation.

Example 7: Rheological Properties

Based on ISO 11137 (Sterilization of health care products—Radiation),radiation sterilization methods can be used with 25 kGy or 15 kGyirradiation as the sterilization dose to achieve a sterility assurancelevel, SAL, of 10⁻⁶.

The rheology results are shown in FIGS. 7 and 8 for PuraStat® with gammairradiation sterilization before and after gelation, respectively. Thedetermined rheological results are listed in Tables 14-15.

TABLE 14 Results from frequency tests of PuraStat ® (RADA16 (SEQ IDNO: 1) 2.5%) with gamma irradiation at 0.1% of strain with 40 mmcone-plate Storage Modulus G′ at 1 Hz (Pa) Gamma- Gamma- Gamma-irradiated irradiated irradiated PuraStat ® PuraStat ® PuraStat ®PuraStat ® Sample # control at 23 kGy at 25 kGy at 40 kGy 1 343.7 514.1546.0 551.0 2 323.3 449.7 490.2 607.7 3 301.7 449.5 467.0 618.4 Mean322.9 471.1* 501.0* 592.4*^(,$) SD 21.0 37.2 40.6 36.2 *denotes if p <0.05 compared to control (two tailed Student's t-test). ^($)denotes if p< 0.05 compared to the others (two tailed Student's t-test).

PuraStat® gamma-irradiated at 23 kGy, 25 kGy, and 40 kGy showed higherstorage modulus than PuraStat® control (FIG. 7 and Table 14). PuraStat®gamma-irradiated at 40 kGy showed significantly higher storage modulusthan PuraState® gamma-irradiated at 23 and 25 kGy. Also, PuraStat®gamma-irradiated at 25 kGy showed slightly higher storage modulus thanPuraStat® gamma-irradiated at 23 kGy.

This indicates that gamma-irradiation positively affected therheological properties of PuraStat®. PuraStat® gamma-irradiated at 23kGy (471.1±37.2 Pa), 25 kGy (501.0±40.6 Pa) and 40 kGy (592.4±36.2 Pa)exhibited 46%, 55%, and 83% increases in their storage moduli,respectively, compared to PuraStat® control (322.9±21.0 Pa).

TABLE 15 Results from frequency tests of PuraStat ® (RADA16 (SEQ IDNO: 1) 2.5%) with gamma irradiation after gelation. Samples were treatedwith DMEM for 20 min at 0.1% of strain with a 40 mm cone-plate StorageModulus G′ at 1 Hz (Pa) Gamma- Gamma- Gamma- irradiated irradiatedirradiated PuraStat ® PuraStat ® PuraStat ® PuraStat ® Sample # controlat 23 kGy at 25 kGy at 40 kGy 1 4454 8866  6885 14568 2 5280 8845 1039510286 3 5183 8622 10148 12134 Mean 4972  8711*  9143*  12330* SD 451 135  1959  2148 *denotes if p < 0.05 compared to control (two tailedStudent's t-test). ^($) denotes if p < 0.05 compared to the others (twotailed Student's t-test).

Furthermore, after gelation triggered by simulated body fluid (i.e.,DMEM buffer) for 20 min, PuraStat® gamma-irradiated at 23 kGy (8711±135Pa), 25 kGy (9143±1959 Pa) and 40 kGy (12330±2148 Pa) exhibited 75%,84%, and 148% increases in their storage moduli, respectively, comparedto PuraStat® control (4972±451 Pa) (FIG. 8 and Table 11).

The rheology results are shown in Tables 16 and 17 for PuraStat® withX-ray irradiation sterilization before and after gelation, respectively.

TABLE 16 Results from frequency tests of PuraStat ® (RADA16 (SEQ IDNO: 1) 2.5%) with X-ray irradiation at 0.1% of strain with 40 mmcone-plate Storage Modulus G′ at 1 Hz (Pa) PuraStat ® X-ray-irradiatedX-ray-irradiated Sample # control PuraStat ® at 25 kGy PuraStat ® at 40kGy 1 343.7 448.3 610.2 2 323.3 429.3 632.5 3 301.7 471.7 578.9 Mean322.9 449.8* 607.2*^(,$) SD 21.0 21.2 27.0 *denotes if p < 0.05 comparedto control (two tailed Student's t-test). ^($)denoted if p < 0.05compared to the others (two tailed Student's t-test).

PuraStat® X-ray-irradiated at 25 kGy and 40 kGy also showed higherstorage modulus than PuraStat® control. PuraStat® X-ray-irradiated at 40kGy showed significantly higher storage modulus that PuraStat®X-ray-irradiated at 25 kGy. This indicates X-ray irradiation positivelyaffected the rheological properties of PuraStat®. PuraStat® irradiatedat 25 kGy (449.8±21.2) and 40 kGy (607.2±27.0 Pa) exhibited 39% and 88%increases in their storage moduli, respectively, compared to PuraStat®control (322.9±21.0 Pa) (Table 17).

TABLE 17 Results from frequency tests of PuraStat ® (RADA16 (SEQ IDNO: 1) 2.5%) with X-ray irradiation after gelation. Samples were treatedwith DMEM for 20 min at 0.1% of strain with a 40 mm cone-plate. StorageModulus G′ at 1 Hz (Pa) PuraStat ® X-ray-irradiated X-ray-irradiatedSample # control PuraStat ® at 25 kGy PuraStat ® at 40 kGy 1 4454 956312832 2 5280 6404 10611 3 5183 9522 10294 Average 4972  8497*  11246* SD451 1812  1383 *denotes if p < 0.05 compared to control (two tailedStudent's t-test).

Furthermore, after gelation triggered by simulated body fluid (i.e.,DMEM buffer) for 20 min, PuraStat® X-ray-irradiated at 25 kGy (8497±1812Pa) and 40 kGy (11246±1383 Pa) exhibited 71% and 126% increases in theirstorage moduli, respectively, compared to PuraStat® control (4972±451Pa) (Table 17).

The rheology results are shown in Tables 18 and 19 for PuraStat® withe-beam irradiation sterilization before and after gelation,respectively.

TABLE 18 Results from frequency tests of PuraStat ® (RADA16 (SEQ IDNO: 1) 2.5%) with e-beam irradiation at 0.1% of strain with 40 mmcone-plate. Storage Modulus G′ at 1 Hz (Pa) PuraStat ® e-beam-irradiatede-beam-irradiated Sample # control PuraStat ® at 25 kGy PuraStat ® at 40kGy 1 343.7 348.1 349.6 2 323.3 356.1 371.5 3 301.7 324.0 338.4 Mean322.9 342.7 353.2 SD 21.0 16.7 16.8

PuraStat® e-beam-irradiated at 25 kGy and 40 kGy, however, did not showa significant change in storage modulus compared to PuraStat® control.This indicates e-beam irradiation sterilization does not have a majoreffect on the rheological properties of PuraStat®. However, although thep values did not show statistical significance (i.e., p>0.05), PuraStat®X-ray-irradiated at 25 kGy (342.7±16.7 Pa) and 40 kGy (353.2±16.8 Pa)exhibited 6% and 9% increases in their storage moduli, respectively,compared to PuraStat® control (322.9±21.0 Pa) (Table 18).

TABLE 19 Results from frequency tests of PuraStat ® (RADA16 (SEQ IDNO: 1) 2.5%) with e-beam irradiation after gelation. Samples weretreated with DMEM for 20 min at 0.1% of strain with 40 mm cone-plate.Storage Modulus G′ at 1 Hz (Pa) PuraStat ® e-beam-irradiatede-beam-irradiated Sample # control PuraStat ® at 25 kGy PuraStat ® at 40kGy 1 4454 3623 4918 2 5280 4830 9327 3 5183 3542 7875 Average 4972 40077373 SD 451 715 2247

After gelation triggered by simulated body fluid (i.e., DMEM buffer) for20 min, PuraStat® e-beam-irradiated at 25 kGy and 40 kGy did not showsignificant difference (i.e., the p values were higher than 0.05) intheir storage moduli compared to PuraStat® control (Table 19).

PuraStat® demonstrated shear thinning at high shear rate and thixotropicbehavior suggesting slow rheological property recovery when highshearing stopped (FIG. 9 ). From these properties of PuraStat®, thestiffness of PuraStat® can be lowered for easier handling duringapplication to patients and stiffness can then slowly recover to initialvalues after application. These intrinsic thixotropic properties ofPuraStat® were not changed, even after gamma irradiation at 23 and 25kGy, while irradiated PuraStat® showed higher storage modulus thanPuraStat® control.

Because the peptides' molecular structure was not substantially changedconsidering the results of HPLC and Mass Spectrometry, the assemblednanofibrous structure of the peptides could be a factor to increasetheir rheological properties. The rheological properties ofself-assembling peptide solution might increase when self-assembledpeptide structure is more organized. By way of a non-binding theory,high energy from irradiation can make peptide molecules move slightly tohave more organized nanofibrous structure resulting in improvedrheological properties.

The increased rheological properties were at least partially reversed byrepeated high shearing and returned back close to the original levels.After thoroughly shearing them twice at 1000 s⁻¹ for 1 minute, PuraStat®samples irradiated at 23 kGy and 25 kGy showed gradual decrease in theirstorage modulus from 484.5 Pa to 410.9 Pa and 514.3 Pa to 426.5 Pa,respectively, while PuraStat® control did not show significant change inits storage modulus (FIG. 9 ). It could be expected that the rheologicalproperties of irradiated PuraStat® become closer to those of PuraStat®control with more shearing. Therefore, gamma irradiation enhances thestructure of self-assembled nanofibers to increase their rheologicalproperties without a detectable change in peptide molecular structuredegradation or crosslinking.

Example 8: Bioburden Testing

A. Collection of Samples. Upon irradiation, 50 samples were collected asfollows: 10 samples for bioburden tests inside the packages (collecting4 at the beginning, each 3 at the middle and the end of the packagingoperation); 10 samples for bioburden tests of the filling liquid in thesyringe (collecting 4 at the beginning, each 3 at the middle and the endof the packaging operation); spare samples, e.g. 30 samples, may becollected in case extra testing may be required.

B. Viable Bacteria Count Test for Sterilization Validation. The samplewas aseptically prepared in a clean bench, and all instruments andsolvents to be used were sterilized.

For blister-packaged products, the following procedure was repeatedtwice with one sealed package to make a total 100 mL of sample solution.A syringe was used to inject 50 mL of rinsing fluid for thesterilization test (USP Fluid D) into the inside of the package. Asample were shaken thoroughly and allowed to stand to reduce bubbles.Then, the outside opening of the package was sterilized by lightlypassing the opening through a flame without heating the contents. Then,a syringe or another suitable method was used to extract all theinjected solution from the package, which was then collected in aheat-resistant bottle. With a sealed blister package containing about100 CPU of spores purified from a standard strain such as Bacillussubtilis (NBRC3134), a recovery rate and a correction coefficient basedon the recovery rate were calculated in advance from the viable bacteriacount and the added bacteria count from a sample which was prepared inthe same manner as above. A correction coefficient was calculated as1/recovery rate (%)×100. This correction coefficient was recalculatedwhen the sample preparation procedure was changed.

For testing content fluid, 1 mL of the content solution was mixed with 9mL of Soybean-Casein Digest Medium agar medium (SCD agar medium), andthe gel finely dispersed to make 10 mL of sample solution.

For blister package products, 100 mL of the sample solution per culturemedium was used and the test was conducted using the membrane filtermethod of the Japanese Pharmacopoeia Microbial Limit Test.

For testing content fluid, 1 mL of the sample solution per culturemedium and conduct the test using the agar plate dilution method of theJapanese Pharmacopoeia Microbial Limit Test. This test was repeated 10times to obtain 10 culture media plates corresponding to 10 mL of samplesolution.

The cultures were maintained at 30° C.-35° C. for 3-5 days (or longer)on SCD agar medium. As a general rule, the cultures were observed atleast once every operating day during the culture period and on thefinal measurement day.

After completion of the culture, the actual measured values of thecolonies of SCD agar medium were converted with the followingcalculation:

-   -   (1) For blister package products        -   Viable bacteria count in a blister package=Viable bacteria            count in 100 mL of sample liquid×Correction coefficient    -   (2) For Content fluid        -   Viable bacteria count in 1 mL of content liquid=Total viable            bacteria count in 10 mL equivalent to sample solution (10            culture media plates)            Remarks: Among the test method for the content fluid, the            value of the SIP (aliquot) used for calculation were            determined to be 1/5=0.2, not the total amount when testing            with a 5-mL product by the test method equivalent to 1 mL of            content liquid. *SIP (Sample Item Portion) is equivalent to            an aliquot (defined portion of the healthcare product used            for testing).

Example 9: Experimental Design of PuraStat® Sterilization by GammaIrradiation

Irradiation conditions—PuraStat® samples (Lot #17C09A30) were irradiatedwith gamma rays at 25, 28 and 40 kGy with Gammacell 220® High Dose RateCo-60 Irradiator (MDS Nordion, Ottawa, Canada). The run dose rate andduration time for 40 kGy irradiation were 6.30 kGy/hr and 6 hours 20minutes 58 seconds, respectively. The run dose rate and duration timefor 28 kGy irradiation were 4.40 kGy/hr and 6 hours 22 minutes 31seconds, respectively. The run dose rate and duration time for 25 kGyirradiation were 6.58 kGy/hr and 3 hours 47 minutes 42 seconds,respectively.

Methods and test results: The appearance of PuraStat® was observed aftereach test. The pH of PuraStat® was tested using an Accumet AB15 pH meter(Fisher Scientific). HPLC tests were performed to evaluate the majorpeptide content after irradiation tests. Agilent HPLC 1100 (AgilentTechnologies) was used for this study. Column temperature was kept at25° C.

Solvent A was water with 0.1% TFA and solvent B was 80% Acetonitrilewith 0.1% TFA. Gradient of solvent B was controlled from 10% to 40% in20 min and 40% for another 5 min at 25° C. Agilent Zorbax 300SB-C18column (4.6 mm×250 mm, 5 m, 300 Å) was used for this test. PuraStat®(RADA16 (SEQ ID NO:1) 2.5%) (40 mg) were mixed with 10 μL of DH₂O and500 μL of formic acid and vortexed. And the mixture was mixed with DH₂O(4,450 μL) and vortexed. 20 μL of samples were injected using an Agilentautosampler.

Mass spectrometry tests were carried out to investigate the degradationof the peptides after irradiation sterilization. Agilent LC/MSD ion trapmass spectrometer was used for this study. The sample solutions wereprepared as described above for the HPLC samples. Each sample wasinjected at 9 μL/min with a syringe pump. Mass spectrum was recorded for1 minute. The molecular weight of PuraStat® (Ac-(RADA)₄-NH₂) (SEQ IDNO:1) is 1712, which matches its calculated molecular weight from themajor three peaks at m/3=572, m/2=857, and m=1713 in all the spectra ofcontrol and irradiated PuraStat® samples.

The assigned peaks to Ac-RADARADARADARADA-NH₂ (SEQ ID NO:1) are,

M/z=572=(Mw+3)/3, so the calculated Mw=1713

M/z=857=(Mw+2)/2, so the calculated Mw=1712

M/z=1713=Mw+1, so the calculated Mw=1712

The rheological properties of PuraStat® samples before and after gammasterilization were evaluated using a rheometer (Discovery HR 1, TAInstruments) at 37° C. Flow tests were carried out with 20 mmplate-plate geometry and 800 μm of gap distance at a shear rate range of0.001 1/sec to 3,000 1/sec at 37° C. Sample solution (350 μL) was placedon the rheometer plate and excess solution was gently removed;measurements were performed after 2 minutes of relaxation time at 37° C.The viscosity (h) was recorded from very low shear rate (0.001 1/sec) tohigh shear rate (3000 1/sec).

L929 neutral red uptake tests were performed to investigate thecytotoxicity of the irradiated PuraStat®. These tests were performed byToxikon located in Bedford Mass., USA. The study was done based upon ISO10993-5, 2009, Biological Evaluation of Medical Devices—Part 5: Testsfor in Vitro Cytotoxicity and ISO 10993-12, 2012, Biological Evaluationof Medical Devices—Part 12: Sample Preparation and Reference Materials.The biological reactivity of a mammalian cell monolayers, L929 mousefibroblasts, in response to the test article extract was determined. Thetest article extract was obtained with serum-supplemented MinimumEssential Medium (MEM) at the ratio 0.2 g of article per mL. Extractionwas done for 24±2 hours at 37±1° C. Positive control (Natural Rubber)and negative control (Negative Control Plastic) articles and anuntreated control (blank) were prepared to verify the proper functioningof the test system. The test article and control article extracts wereused to replace the maintenance medium of the cell culture. The testarticle extract was tested at the 100% (neat) concentration. Allcultures were incubated in at least 6 replicates for 24 to 26 hours at37±1° C., in a humidified atmosphere containing 5±1% carbon dioxide(C02). The viability of cells following the exposure to the extracts wasmeasured via their capacity to uptake a vital dye, Neutral Red. This dyewas added to the cells to be actively incorporated in viable cells. Thenumber of viable cells correlates to the color intensity determined byphotometric measurements at 540 nm after extraction.

The viability of cells exposed to the negative control article andpositive control article extracts needs to be greater than or equal to70% and less than 70% of the untreated control, respectively, to confirmthe validity of the assay. The test article meets the requirements ofthe test if the viability % is greater than or equal to 70% of theuntreated control.

We tested only PuraStat® samples irradiated at the highest dose (i.e.,40 kGy) considering they represent all the irradiated samples, becausethey should have more effect, if any, on cytotoxicity than thoseirradiated at lower doses.

TABLE 20 Summary Table of Irradiation Sterilization Tests Testing HPLCCytotox- conditions Appearance pH and MS Rheology icity PuraStat ® Clearand 2.2 Control Control Not toxic (Control) viscous PuraStat ® withClear and 2.3 ~5% — — Gamma at 25 kGy viscous degrade PuraStat ® withClear and 2.3 ~15% equivalent Not toxic Gamma at 28k or viscous degrade40 kGy

The amount of peptide prior to the irradiation. After up to 25 kGy ofirradiation, PuraStat®'s degradation did not exceed 5% of the amount ofpeptide prior to the irradiation.

From the pattern of degradation of control PuraStat®, we found thatdegradation mainly occurs at the points between ˜RAD and A˜. From theadditional pattern of degradation of Ac-(RADA)₄-NH₂ (SEQ ID NO:1) whenPuraStat® is sterilized with irradiation, we found that irradiation cancause additional degradation at the point between ˜R and AD˜.

Overall, the rheological property of PuraStat® with gamma irradiationwas equivalent to PuraStat® control. All the irradiated PuraStat® andPuraStat® control samples meet the requirements of the test and are notconsidered to have a cytotoxic potential.

Table 21 below shows results of HPLC tests was performed to evaluate themajor peptide content of PuraStat® before (control) and after Gammairradiation.

TABLE 21 The HPLC and LC-MS analysis for two Product Form HPLC and LCMSresults in each peak PuraStat ® current product (non-irradiated)PuraStat ® proposed product (irradiated) HPLC LC-MS HPLC LC-MS Repre-Repre- Area sentative Area sentative Peak t_(R) Area % m/z Peak t_(R)Area % m/z 1 6.37 25.70 4.39 1329(666) 1 6.35 8.20 1.90 1329(666) 2 6.738.49 1.44 1229(615) 2 6.73 2.90 0.67 1229(615) 3 7.15 9.98 1.711300(651) 3 7.27 12.00 2.78 1143(572) 4 7.30 9.29 1.59 1143(572) 4 7.562.29 0.53 1671(836) 893 5 7.75 4.75 0.81  1671(836), 5 7.71 2.34 0.541671(836) 1713(857) 6 8.01 7.49 1.28  1713(857), 6 7.90 7.62 1.77 1671(836), 750 1713(857) 7 8.18 6.95 1.19  1643(823), 7 8.16 4.06 0.94 1643(823), 1713(857)  1713(557), 1513 8 8.29 2.15 0.36  1643(823), 88.27 7.95 1.84  1643(823), 1713(857)  1713(557), 1513 9 8.45 473.2380.80 1713(857) 9 8.47 348.78 80.89  1713(857) 10 8.80 11.89 2.03 1557,783, 857 10 8.77 12.95 3.00 1557, 783 11 8.93 4.54 0.77 783, 857 11 129.06 4.85 0.82 839, 12 9.00 10.46 2.43 839, 1713(857)  1713(857) 1310.00 16.26 2.77 482, 839, 891 13 9.96 11.61 2.69 839, 482

Agilent HPLC 1100 (Agilent Technologies) was used for this study. Columntemperature was kept at 25° C. Agilent Zorbax 300SB-C18 column (4.6mm×250 mm, 5 mm, 300 Å) was used for this test. Solvent A was water with0.1% TFA and solvent B was 80% Acetonitrile with 0.1% TFA. Gradient ofsolvent B was controlled from 10% to 40% in 20 min and 40% for another 5min at 25° C. PuraStat® (40 mg) was mixed with 10 μL of DH₂O and 500 μLof formic acid and vortexed, and the mixture was mixed with DH₂O (4,450μL) and vortexed. 20 mL of samples were injected using an Agilentautosampler. Results are shown in following Table 23.

TABLE 23 PuraStat ® PuraStat ® irradiated control above 25 kGy MajorPeptide 83.8 +/− 1% 81.3 +/− 0.7% Content (n = 3) Manufacturing 75% andmore Specification

Findings—Overall, the major peptide content of PuraStat® decreased withgamma irradiation methods and the extent of decrease was moresignificant with higher dose. After up to 40 kGy of irradiation,PuraStat®'s degradation did not exceed 15% of the amount of peptideprior to the irradiation. After up to 25 kGy of irradiation, PuraStat®'sdegradation did not exceed 5% of the amount of peptide prior to theirradiation.

From the pattern of degradation of control PuraStat®, we found thatdegradation mainly occurs at the points between ˜RAD and A˜. From theadditional pattern of degradation of Ac-(RADA)₄-NH₂ (SEQ ID NO:1) whenPuraStat® is sterilized with irradiation, we found that irradiation cancause additional degradation at the point between ˜R and AD˜.

Overall, the rheological property of PuraStat® with gamma irradiationwas equivalent to PuraStat® control. All the irradiated PuraStat® andPuraStat® control samples met the requirements of the test and were notconsidered to have a cytotoxic potential.

The invention claimed is:
 1. A sterilized solution of self-assemblingpeptide (SAP) made by sterilizing a self-assembling peptide (SAP)solution, the method of sterilization comprising: (a) placing one ormore containers with the solution of self-assembling peptide into anirradiation machine, said self-assembling peptide selected from thegroup consisting of RADA16 (SEQ ID NO:1), KLD12 (SEQ ID NO:2), andIEIK13 (SEQ ID NO:3), and capable of forming a hydrogel when applied toa biological tissue at about neutral pH; and (b) exposing the one ormore containers to gamma ray, X-ray, and/or e-beam irradiation at apredetermined dose so that the self-assembling peptide solution issterilized to a pre-determined Sterility Assurance Level (SAL) withoutsubstantial degradation of the peptide wherein the concentration of thedegradation products of a full-length peptide in the solutionpost-irradiation ranges from 0.1% to 5% w/v while its desired biologicalor physical properties are maintained substantially at the same level orimproved; wherein the desired biological or physical properties areselected from the group consisting of: hemostatic, anti-adhesion,prevention of re-bleeding, anti-stenosis, tissue occlusion, mucosaelevation, wound healing, storage modulus, viscosity, and tissue voidfilling property.
 2. The self-assembling peptide solution of claim 1,wherein the dose is 15-50 kGy.
 3. The self-assembling peptide solutionof claim 2, wherein the solution is irradiated by X-ray or e-beam. 4.The self-assembling peptide solution of claim 1, wherein the peptidesolution contains about 2.5% w/v of RADA16 (SEQ ID NO:1), and the doseis 15-24 kGy.
 5. The self-assembling peptide solution of claim 1,wherein the amount of total peptides that are degraded after theirradiation does not exceed 20% by weight of the amount of theself-assembling peptide prior to the irradiation.
 6. The self-assemblingpeptide solution of claim 5, wherein the pre-irradiation bioburden ofthe self-assembling peptide solution is 9 CFU or less.
 7. Theself-assembling peptide solution of claim 1, wherein irradiation doseachieves sterility assurance level (SAL) of at least 10⁻⁶.
 8. Theself-assembling peptide solution of claim 1, wherein the pH of thesolution pre- and post-irradiation ranges from about 1.8 to 3.5.
 9. Theself-assembling peptide solution of claim 1, wherein the one or morecontainers is/are a plastic syringe(s).
 10. The self-assembling peptidesolution of claim 1, wherein the storage modulus of the gelled solutionis increased at least by 10% post-irradiation.
 11. The self-assemblingpeptide solution of claim 1, wherein the self-assembling peptidesolution is subjected to shearing post-irradiation to reduce or restoreits storage modulus.
 12. The self-assembling peptide solution of claim1, wherein the SAP solution exhibits a post-irradiation massspectrometric (MS) profile having major M_(z) peaks at 836/1670, 1100,and 1513 m/z.